The present invention relates to transformed plants obtained by constructing recombinant DNAs such as sense genes or antisense genes using CDNA that encodes 4-coumarate: coenzyme A ligase (hereinafter referred to as 4CL), an enzyme occurring in plants, and then introducing them into the genome to obtain the transformed plants.
In general, major building blocks of the cell wall of higher plants include, for example, lignin, a polymer compound having an aromatic nucleus, in addition to polysaccharides such as cellulose and hemicellulose. Lignin is known to deposit on the cell wall by the polymerization of cinnamyl alcohols biosynthesized from an essential amino acid phenylalanine of a living body via a plurality of enzymatic reactions. Lignin synthesized in the cell serves, in a sense, as a reinforcement for the maintenance of plant morphology together with polysaccharides, and it is being elucidated that it also plays an important role in the resistance to various stresses.
However, the presence of lignin, which is important for the maintenance of biological functions of plants, is not always welcome when materials in the plants are to be utilized as raw materials in various industries. In the pulping and paper making industry, for example, wood chips, construction waste wood, or unused residues derived from herbaceous plants and the like are used to produce pulp that makes a raw material for paper making. However, in many processes for pulp production, considerable amounts of energy and chemicals are used to remove lignin from wood materials. In the dairy industry too, grass and various plant residues are in common use as the fodder. In cases where the fodder taken up by domestic animals is digested in the digestive tract, it is known, lignin present in the fodder hinders its digestibility thereby reducing the efficiency of the fodder.
As one of the means to solve the above industrial problems associated with the presence of lignin, attempts have been made in the last few years to modulate the amount of lignin deposited on plant cell walls or the molecular structure of lignin using genetic manipulation. These attempts are intended to modify the functions for genes of enzymes involved in the biosynthesis of lignin to alter the amount of lignin formed in the cell wall and to alter the molecular structure of lignin. By facilitating changes in lignin biosynthesis using these methods, it is expected to be able to develop plants that have industrially advantageous properties.
For example, attention has been given to phenylalanine ammonia-lyase, an enzyme that catalyzes the initial reaction for synthesis of phenylpropanoid including lignin; in a transformed tabacco wherein the expression of the gene has been suppressed, it is stated, lignin content in plants is reduced and the relative amount of guaiacyl lignin is enhanced to that of syringyl lignin (Sewalt et al., Plant Physiology, vol 115, 41, 1997).
It has been reported that in a transformed Arabidopsis which expresses at a high level the gene of ferulate 5-hydroxylase, an enzyme thought to play an important role in the control of the relative amount of syringyl lignin in the entire lignin structure, the relative amount of syringyl lignin is enhanced (Chapple et al., Proc. Natl. Acad. Sci. U.S.A., vol 95, 6619, 1998).
U.S. Pat. No. 5,451,514 discloses, in an experiment employing transformed tabacco, that suppressing the expression of the gene of cinnamyl alcohol dehydrogenase (hereinafter referred to as CAD) involved in lignin monomer biosynthesis results in the modification of biosynthetic processes of phenolic compounds including lignin, thereby increasing the amount of lignin extracted from the cell wall associated with alkali treatment.
Furthermore, U.S. Pat. No. 5,850,020 and U.S. Pat. No. 5,952,486 disclose methods of reducing the lignin content in recombinant tabacco relative to that in the wild type plant, which method comprises isolating cDNAs of a plurality of enzymes related to lignin biosynthesis obtained from Pinus radiata and Eucalyptus grandis, and introducing recombinant DNA that uses these cDNAs into the genome of tabacco.
In addition to the above examples, the inventors of the present invention have disclosed in Japanese Unexamined Patent Publication (Kokai) No. 9-173069 that suppressing the expression of the 4CL gene that is a target gene of the present invention results in reduced lignin content in transformed tabacco and significant changes in the molecular structure of lignin. It has also been shown, however, that extremely abnormal growth occurs at a high frequency in transformed tabacco in which the expression of the gene has been suppressed (Kajita et al., 1996, Plant and Cell Physiology, 37, 957). If the modification of lignin in plants obtained by suppressing the expression of the 4CL gene is useful for industrial application, recombinant plants having such properties could develop an extremely abnormal growth, which would reduce the production efficiency of the plants themselves, and thereby, it is expected, would increase the total cost required in the processes from the cultivation of said plants to the production of final products. And therefore, their application into the industry in the art would be limited.
WO 99/24561 discloses a method which comprises suppressing the activity of the 4CL gene in aspen, thereby reducing the content of lignin present in the cell wall of the transformed aspen, and at the same time modifying the growth characteristics of the transformed aspen. It shows a similar effect to that of the present invention in that it reduced the lignin content in the cell wall, but it makes no mention of the characteristics of pulp production for wood chips obtained from the recombinant aspen.
On the other hand, Petit-Conil et al. conducted a pulping test using a transformed poplar in which the expression of the CAD gene had been suppressed. The result reveals that by using wood chips obtained from said transformed poplar, delignification is easier under the same condition than when using those obtained from the wild type plants (Proceedings of the 1998 7th International Conference on Biotechnology in the Pulp and Paper Industry, A37-A41, 1998). Although the result indicates that it is possible to obtain chip materials having excellent pulp productivity by modifying the function of the gene of enzymes involved in lignin monomer biosynthesis, the CAD gene noted by Petit-Conil et al. is entirely different from the 4CL gene noted in the present invention.
Thus, there has not been obtained at present a method of improving the characteristics as a raw material for pulp retained by woody substances containing lignin obtained from plants by modifying the expression of the endogenous 4CL gene to plants, and thereby modifying the lignin content or the molecular structure of lignin present in the plants.
It is an object of the present invention to provide transformed plants that offer woody substances more suitable for pulp production by modifying the content and the molecular structure of lignin deposited in the cell wall by modifying the expression of the 4CL gene.
As a result of intensive research in order to solve the above problems, the inventors of the present invention have successfully created transformed plants that exhibited decreased levels of 4CL activity. Furthermore, we have proved, based on the result of the pulping experiment using wood chips derived from these transformed plants, that the amount of pulp obtained from said plants is significantly high compared to that obtained from wild type plants when a delignification treatment was carried out to a similar degree to these plants, and thereby have completed the present invention.
Thus, the present invention relates to transformed plants that contribute to enhancing pulp productivity, and more specifically it relates to (1) CDNA of 4-coumarate:coenzyme A ligase having the nucleotide sequence as set forth in SEQ ID NO: 1, (2) CDNA of 4-coumarate:coenzyme A ligase having the nucleotide sequence as set forth in SEQ ID NO: 2, (3) a recombinant DNA having at least a portion of these cDNAs inserted in an antisense orientation to DNA containing any promoter and terminator sites, (4) a recombinant DNA having at least a portion of these CDNAs inserted in a sense direction to DNA containing any promoter and terminator sites, (5) a transformed plant obtained by introducing these recombinant DNAs into the genome, (6) a transformed plant in which the plant is a woody plant, (7) a transformed plant in which the woody plant is a plant of genus Eucalyptus, (8) cells obtained from these transformed plants, (9) seeds obtained from these transformed plants, (10) fruits obtained from these transformed plants, (11) a method of introducing the above recombinant DNA into the genome to change the production level of enzymes having a 4-coumarate:coenzyme A ligase activity, thereby changing the content of lignin present in the cell wall of the transformed plants, and (12) a method of introducing the above recombinant DNA into the genome to change the production level of enzymes having a 4-coumarate:coenzyme A ligase activity, thereby changing the molecular structure of lignin present in the cell wall of the transformed plants.